This project concerns the death of cells through the process called apoptosis, which is critical both for normal physiology and development. Apoptosis is also important for the origin and treatment of various diseases, including cancer, AIDS, and disorders of the immune system and the central nervous system. A basic understanding of the apoptotic cell death machinery will help us understand both normal cellular physiology and also provide a foundation for developing better disease therapies. In this project, we are interested in how mitochondria play a key role in apoptotic cell death and how this cell death function is controlled by proteins belonging to the Bcl-2 family. In particular, we want to understand how one of these proteins, Bax, forms pores in the mitochondrial outer membrane. Permeabilization of the outer membrane allows the release of toxic proteins that trigger the apoptotic process. The end result is that cells are actively dismantled and then consumed and destroyed by other cells called phagocytes. Our studies will use cell-free systems we have developed, using either artificial membranes or isolated outer mitochondrial membranes, to help uncover the basic mechanisms through which Bax forms membrane pores. Other studies will use NMR spectroscopy to examine structural changes in the Bax molecule produced by certain compounds that inhibit or promote Bax pore formation. We will also examine the communication that occurs between Bax, at the outer mitochondrial membrane and another protein called Opa1, in the inner mitochondrial membrane. We also want to understand how the linkage between these proteins may relate to a genetic disease, Autosomal Dominant Optic Atrophy, which is caused by mutations in Opa1. PUBLIC HEALTH RELEVANCE: Cells often die by a preset internal program called apoptosis. Apoptotic cell death is important in both normal human development, in sculpting tissues and preventing the accumulation of excess cells. Apoptosis is also important in normal adult organs and the immune system, to keep cell populations at normal sizes. If apoptotic cell death is abnormally controlled, cells can either die inappropriately, leading to a loss of essential cell populations, or can survive and proliferate to an unhealthy level. Abnormal apoptosis can be involved in such diseases such as cancer, AIDS, and autoimmune or neurodegenerative disorders. Furthermore, apoptotic death of cancer cells is an important component of cancer therapy. For all these disease states, it is important to understand the basic processes involved in cell death, not only to understand disease origins, but also to help devise more effective treatments. If this project is funded, we want to uncover the ways in which mitochondria, energy-producing structures inside cells, are critical for various processes that lead to cell death. In particular, we are interested in a family of proteins related to a pro-cancer protein, Bcl-2; especially with regard to how these proteins control the formation of pores in the membrane surrounding each mitochondrion. This pore formation allows toxic proteins to escape, leading to cell death. Secondly, we want to understand how another protein, Opa1, functions normally within mitochondria to keep cells. We also want to understand how mutations of Opa1 that occur in patients with a disease called Autosomal Dominant Optic Atrophy can cause hereditary loss of vision later in life.